1. Field of Invention
Aspects of the invention relate to a method of immobilizing molecules onto a solid phase substrate, especially to method of immobilizing a plurality of molecules to a single substrate, a method of fabricating a biosensor using the molecule immobilization method, and method of detecting a target material.
2. Description of Related Art
Related biosensors with molecules having affinity with target materials and fixed on solid phase substrates are becoming in widely spread use for detecting the target materials included in test samples. These related biosensors utilize mutual interactions (hybridizations) between complementary nucleic acid strands, enzymes and stromas, enzymes and coenzymes, antigens and antibodies, or other mutual interactions between accepters and their ligands to capture target materials on surfaces of solid phase substrates, thereby determining directly or indirectly (e.g., determination using fluorescent molecules combined with the target materials) presence of the target materials. Therefore, the probes are often biomolecules, such as nucleic acids, proteins, or sugars.
Methods of immobilizing these biomolecules onto substrates are categorized mainly into methods (e.g., nucleic acid elongation reaction) of composing these molecules directly on solid phase substrates and methods of immobilizing previously composed molecules to surfaces of substrates. As an example of the latter methods, a method can be used in which a specific group is coupled, via a linker, to the tail end of a molecule to be immobilized and the molecule is absorbed onto a solid phase substrate by incubating a solution including the molecule with contacting to the substrate. The absorption method is particularly used in case of immobilizing a molecule difficult to be composed on a substrate or when immobilizing a number of kinds of different molecules onto a single substrate.
In immobilizing probe molecules by the absorption method, if the pitch of the probes is too narrow, relatively large, bulky target molecules, such as DNA or proteins, may interfere each other with coupling with the probe. Accordingly, in the absorption method, in order to control the density of the probes so that as many probe molecules and target molecules as possible are effectively be coupled, relatively small molecules called spacer is generally added to the probe molecule solution to be immobilized simultaneously.
Meanwhile, as a related biosensor which immobilizes enzymes as probes and utilizes specificity of enzyme-stroma reactions, for instance, a sensor which immobilizes glucose oxidases to measure glucoses in a test sample is known and is under development also as a blood sugar level measuring instrument for home use. Although in glucose sensors, electrode activation materials, such as oxygen or hydrogen peroxide, are generally monitored by electrodes around the time of mutual interactions between glucoses and glucose oxidases, since an amount of dissolved oxygen is not sufficient in case of blood as a test sample. A related method using an electronic mediator, which is a charge transfer complex functioning substitute for oxygen can be used. See, for example, Japanese Unexamined Patent Publication No. 5-203608. Accordingly, in glucose sensors, if both of glucose oxidases and electronic mediator molecules are immobilized in a single solid phase substrate, glucoses can effectively be detected.
In order to immobilize on a single substrate a number of kinds of molecules, such as nucleic acid molecules and spacer molecules or enzymes and enzyme mediators, a coabsorption method can be used. In the coabsorption method, a solid phase substrate can be dipped in a solution containing mixture of a number of kinds of molecules and an amount of molecules absorbed to the solid phase substrate is adjusted by altering the mixing ratio of the molecules, thus enabling, in theory, a density of immobilized probe in the biosensor to be controlled only by initially adjusting the composing ratio of the mixed solution. In view of application to diagnosis or safety tests of foods, biosensors capable of detecting a number of materials included in a test sample at a time are required, and the coabsorption method is hoped to be a technology capable of immobilizing a number of kinds of probe molecules to a solid phase substrate at a time.